The rapid determination of trace metals in biological and environmental systems is increasingly important in identifying potential hazards and preserving the public health. The toxicity of certain metals such as lead is well-known. The absorption of even trace amounts of lead can cause severe damage to human organs. The numerous and widespread sources of lead in the environment, including the food supply, compounds the problems of screening affected groups.
It is generally recognized that lead poisoning occurs in children at blood levels as low as 10-15 ug/dl. Lead contamination of environmental sources such as water, dust and soil require identification at even lower levels. To measure these amounts, the analytical techniques must be sensitive, contaminant-specific, and reliable.
The use of d-aminolevulinic acid dehydratase (ALAD) activity in red blood cells to determine exposure to environmental lead is described by A. Berlin, et al., "European Standardized Method for the Determination of d-Aminolevulinic Acid Dehydratase Activity in Blood," Z. Klin. Chem. Klin. Biochem., 12 Jg. 1974, S. 389-390. The assay entails incubation of the enzyme with excess d-aminolevulinic acid (ALA). The porphobilinogen (PBG) which is formed within a predetermined time interval is mixed with modified Ehrlich's reagent, and the color developed is measured photometrically against a blank. The quantity of PBG produced is a measurement of the ALAD activity corresponds to low levels of lead exposure.
The method of colorimetric determination of ALAD was originally disclosed by K. D. Gibson, et al., "The Purification and Properties of d-Aminolaevulic Acid Dehydrase," Bioch., 61:618-629 (1955). In addition to a purification method, this reference discloses the activation of the ALAD by thiol reagents and the inhibition of the dehydrase activity by copper, mercury and silver.
Subsequently, a modified colorimetric method for lead detection was presented by S. Sassa, "Delta-Aminolevulinic Acid Dehydratase Assay," Enzyme, 28:133-145 (1982). As part of the assay disclosed, the mercury containing compound HgCl.sub.2 is used with trichloroacetic acid (TCA) to precipitate proteins as well as in the modified Ehrlich's reagent to eliminate interference with chromophore formation by sulfhydryl compounds. The reduction effect of sulfhydryl compounds like dithiothreitol (DTT), mercaptoethanol, cysteine and reduced glutathione to increase enzyme activity is also disclosed. The instability of the sulfhydryl compounds require special preparation for their use in the assay.
Two articles, each entitled "Purification and Properties of d-Aminolevulinate Dehydrase from Human Erythrocytes," first published by P. Anderson, et al., J. Biol. Chem., 254:6924-6930 (1979) and subsequently by P. Gibbs, et al., Biochem J., 230:25-34 (1985), disclose assays demonstrating lead as a noncompetitive inhibitor of ALAD activity. The incubation mixtures contained DTT, ALAD and ALA in a buffer solution. The incubations were terminated by the addition of TCA which also contained HgCl.sub.2. The solution was centrifuged and the supernatant was added to modified Ehrlich's reagent in acetic acid and perchloric acid. The colored complex formed with PBG was measured spectrophotometrically.
A similar assay measuring the activity of ALAD after exposure to lead containing samples is disclosed in a published PCT application WO 93/01310 to Silbergeld. The application suggests utilizing other well-known methods like conjugating or attaching a label to either the substrate or product and quantifying the amount of labeled material present after a defined reaction period. Another approach suggested, uses a known antibody that binds specifically to unoccupied lead binding sites of ALAD.
A significant problem in using an ALAD assay is the toxicity of the mercury used in the TCA solution as well as in the modified Ehrlich's reagent to eliminate interference with chromophore formation by sulfhydryl compounds. The disposal of mercury-containing waste products also causes an environmental problem which increases the relative expense of using the assay.
Accordingly, there is a need to replace mercury with a reagent which is less toxic and environmentally hazardous without diminishing the sensitivity and accuracy of the assay. The present invention provides selected metal ions used as colorimetric enhancing reagents to replace mercury in the assays.
Another solution to the problem is to eliminate using mercury altogether. One inventive approach is to replace the prior art sulfhydryl compounds with a reagent which can increase an enzyme's activity without requiring a metal ion like mercury for its effective use.
Although phosphines have been disclosed for reducing proteins, their utility has been limited because they are malodorous, insoluble in water, and those with low molecular weights easily autoxidate. For example, tertiary phosphines are disclosed as reductive agents to cleave gamma-globulin in M. E. Levison, et al., "Reduction of Biological Substances by Water-Soluble Phosphines: Gamma-Globulin (IgG)," Experientia, 25:126-7 (1969). A tertiary phosphine is disclosed by T. L. Kirley, "Reduction and Fluorescent Labeling of Cyst(e)ine-Containing Proteins for Subsequent Structural Analyses," Analytical Biochemistry, 180:231-236 (1989) as part of the process to reduce and alkylate proteins. Furthermore, as disclosed by J. A. Burns, et al., "Selective Reduction of Disulfides by Tris(2-carboxyethyl) phosphine" J. Org. Chem., 56:2648-2650 (1991), qualitative studies of the relative reactivity of the tertiary phosphine with lipoic acid and 2-hydroxyethyl disulfide indicated that the reductions are kinetically, not thermodynamically, controlled.
The present invention provides selected water-soluble tertiary phosphines used as activating reagents to improve the sensitivity and accuracy of a lead assay. The inventive activating reagents replace the prior art sulfhydryl compounds and eliminate using mercury in the assays. In some assays, an inventive activating reagent can be used in combination with an inventive colorimetric enhancing reagent for further improvement of the assay's sensitivity and accuracy.